The juxtaposition of variability and stability in the HYP effectors of globally important plant-parasites.

植物寄生线虫威胁着当前和未来的全球粮食安全，估计每年给世界农业造成的损失超过1000亿美元。在英国，最重要的经济物种是“马铃薯胞囊线虫”，每年给马铃薯产业造成超过5000万GB的损失。鉴于马铃薯孢囊线虫已经存在于世界上每个主要的马铃薯种植区，包括新兴市场，可以提出一个强有力的理由来探索任何可能导致新的控制措施的寄生虫生物学领域。寄生生物学越来越感兴趣的一个领域是“效应器”--线虫衍生的分子注入植物以引起疾病。在这项提议中，我们将集中精力研究一组不寻常的效应器，称为“HYP”，主要原因有两个：首先，HYP背后的遗传学是令人着迷的；其次，干扰HYP可能导致强大的线虫控制。了解HYP的不同寻常的遗传学：到目前为止，所有75个独特的HYP基因共享两个连续的编码序列，基因之间几乎相同：这些“保守区”加在一起，约占基因总长度的一半。HYPs值得注意的是，在这两个近乎相同的保守区之间存在一个“高可变结构域”。这种高度可变的结构域可以编码几个可变序列和组织的“基序”，几乎没有可观察到的模式。更复杂的是，没有一个单独的线虫基因组编码所有的HYP，也没有两个被测试的个体编码相同的HYP。就我们所知，目前还没有已知的机制可以同时解释高度可变的结构域组织和同一种群姐妹之间的基因数量差异。要了解HYP遗传可变性和稳定性的组合，我们需要知道HYP在个体基因组中的位置，以及它们是如何排列的。为了做到这一点，我们将利用最新的技术进步来对单个线虫的含有HYP的超长DNA分子进行测序。虽然我们不能确定是否有任何两个分子来自同一个人(即使它们是相同的)，但我们可以绝对确定每个读数来自一个人。我们可以使用这些超长的DNA序列，结合高质量的群体共识基因组，来探索HYP遗传变异和稳定性的遗传基础。扰乱HYP的功能：我们知道HYP基因的序列是“进化受限的”。含有HYP效应器的物种在大约3000万年前彼此分化，但从那时起，它们的HYP基因的保守区几乎保持不变。顺理成章的是，尽管可变区具有巨大的多样性，但为什么核苷酸序列如此高度保守，肯定有某种解释。考虑到进化对HYP的限制，针对所有马铃薯胞囊线虫物种中所有HYP基因的保守区，可能会产生强大、持久和广谱的抗性。为了破坏HYP的功能，我们建议使用一种称为RNA干扰的靶向方法。RNA干扰可以根据基因的序列扰乱其功能。通过将RNA干扰定位到几乎相同的保守区，我们可以扰乱所有马铃薯胞囊线虫中所有HYP基因的功能。为了在感染期间向线虫传递RNA干扰，我们将创建转基因马铃薯植株，编码针对线虫HYP的RNA干扰基因。当线虫以植物为食时，它们会吃掉干扰的RNA，它们相应的HYP基因就会被破坏。因此，这一提议将有趣的纯科学问题(关于这个系统的基因组进化)与真正的农业影响潜力结合在一起。

Plant-parasitic nematodes threaten current and future global food security, and are estimated to cost world agriculture over $100 billion per year. In the UK, the most economically important species are the "potato cyst nematodes", which cause over £50 million of damages to the potato industry each year. Given that potato cyst nematodes are already present in every major potato growing region of the world, including emerging markets, a strong case can be made for exploring any area of parasite biology that may lead to new control measures. One area of parasite biology of increasing interest is "effectors" - nematode-derived molecules injected into the plant to cause disease.In this proposal we will focus our efforts on an unusual group of effectors called "HYPs", for two main reasons: Firstly, the genetics underlying HYPs are academically fascinating; Secondly, disrupting HYPs could lead to robust nematode control.Understanding the unusual genetics of HYP effectors: All of the 75 unique HYP genes identified to date share two continuous strings of coding sequence that are near identical between genes: together, these "conserved regions" make up approximately half of the total gene length. What is remarkable about HYPs is that between these two near-identical conserved regions lies a "hyper-variable domain". This hyper-variable domain can encode several "motifs" of variable sequence and organisation with almost no observable patterns. To add to the complexity, no one individual nematode genome encodes all HYPs, and no two individuals tested encode the same repertoire of HYPs. To the best of our knowledge, there is no known mechanism that can account for both the hyper-variable domain organisations and the gene number variation between sisters of the same population.To understand the combination of HYP genetic variability and stability, we need to know where HYPs are in the genomes of individuals, and how they are arranged. To do this we will use recent advances in technology to sequence HYP-containing ultra-long DNA molecules from individual nematodes. While we will not be able to determine whether any two molecules came from the same individual (even if they are identical), we can be absolutely certain that each read came from an individual. We can use these ultra-long DNA sequences, in combination with a high-quality consensus genome of the population, to explore the genetic basis of HYP genetic variability and stability.Disrupting HYP function: We know that the sequence of HYP genes are "evolutionarily constrained". The species which contain HYP effectors diverged from one another approximately 30 million years ago, and yet, since that time, the conserved regions of their HYP genes have remained almost unchanged. It stands to reason that there must be some explanation why the nucleotide sequence is so highly conserved, in spite of the tremendous diversity of the variable domain. Given the evolutionary constraint on HYPs, targeting the conserved regions in all HYP genes, in all potato cyst nematode species, may produce strong, durable, and broad spectrum resistance.To disrupt HYP function we propose to use a targeted approach called RNA interference. RNA interference can disrupt the function of a gene based on its sequence. By targeting RNA interference to the near-identical conserved regions, we can disrupt the function of all HYP genes, in all potato cyst nematodes. To deliver RNA interference to nematodes during infection, we will create transgenic potato plants that encode an RNA interference gene which targets the nematode HYPs. When the nematode feeds on the plant, they eat the interfering RNA, and their corresponding HYP genes are disrupted.This proposal thus combines interesting how/what/why pure science questions (about the genomic evolution of this system) with real potential for an agricultural impact.